Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association.
The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.
Matsui
T
T
Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
Maeda
M
M
Doi
Y
Y
Yonemura
S
S
Amano
M
M
Kaibuchi
K
K
Tsukita
S
S
Tsukita
S
S
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D054460:rho-Associated Kinases; D000099149:Ezrin
D000199:Actins; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011994:Recombinant Proteins; D054460:rho-Associated Kinases; D000099149:Ezrin; C071058:moesin; C071776:radixin